Project Summary ? TR&D 2 The Resource has the overall goal of broadly impacting biomedical research by providing the abilities to: obtain high quality proteomics data from much smaller samples, produce more quantitative and comprehensive measurements, generate improved and more extensive information on low abundance components, distinguish presently problematic peptide isomers, and enable the study of much larger sample sets than presently practical by providing increases in measurement sensitivity and throughput. The technology efforts of TR&D 2 will enable these capabilities by providing high resolution separations and ion manipulations with ion mobility- mass spectrometry that result in proteomics measurements with much greater sensitivity and throughput, which in conjunction with the `front-end' developments under TR&D 1, will provide the basis for broadly effective applications of proteomics from much smaller samples. TR&D 2 will develop new platforms based on our novel Structures for Lossless Ion Manipulations (SLIM) technology. These platforms will provide ultra-fast and ultra-high resolution ion mobility (IM) separations that will complement the distinctive advantages provided by both fast time-of flight MS and ultra-high mass accuracy and resolution FTMS (specifically Orbitrap-based MS) platforms. This work will benefit from the inherently fast, robust, and highly reproducible nature of ultra-high resolution IM separations, as well as the distinctive peptide/protein size/shape information (collision cross sections) provided by IM. In combination with the bioinformatics developments of TR&D 3, TR&D 2 will enable improved quantification and proteome coverage in conjunction with high throughput measurements. Overall, TR&D 2 will enable: (1) much more extensive proteomic information from extremely small samples (e.g. from Fluorescence-activated cell sorting and laser capture microdissection) when applied in conjunction with the developments of TR&D 1; (2) large gains in proteomic throughput (measurement speeds) based upon fast ultrahigh-resolution IM separations and related MS-based advances; (3) more quantitative and higher data quality measurements that effectively provide a convergence of the attractive properties of global and targeted MS-based proteomic measurements; and (4) broader proteome measurement coverage that also addresses important `blind spots' of current technologies, such as presently indistinguishable PTMs and other peptide isomers. These efforts will build upon previous Resource developments and will be facilitated by key computational and bioinformatics tool developments under TR&D 3. In combination, these efforts will provide a basis for rapid implementation and initial evaluation of the new capabilities in a set of challenging biomedical projects, as well as their effective dissemination to the research community, including seeding the technology in several outside laboratories and facilitating their initial commercialization, so as to assure broad and sustained availability.